Astrocytes in the brain take up glutamate released by neurons, metabolize glutamate and ammonia to form glutamine, and export glutamine to the extracellular neuronal microenvironment. Of these processes, the export of glutamine is least well understood. Our preliminary studies of murine astrocytes in primary culture show that a widespread transport activity identified functionally as System ASC is the likely mediator of glutamine export. Three cloned transporters (ASCT1, ASCT2, ATB0), related to the glutamate transporter family, are known to exhibit System ASC-like properties, but the substrate specificities of ASCT1 and ATB0 clearly differ from System ASC-mediated glutamine transport in astrocytes. Other preliminary data from our laboratories show that ASCT2 transcripts are abundant in astrocyte cultures. We therefore hypothesize that the ASCT2 transporter mediates glutamine export from astrocytes. In this role, ASCT2 creates a novel linkage between glutamine export and glutamatergic neurotransmission. This linkage occurs because ASCT2 mediates obligatory exchange between glutamine and extracellular L-alanine, L-serine and D-serine, which are endogenous agonists at the glycine modulatory site on the N-methyl-D-aspartate subtype of glutamate receptors. The rate of glutamine export thus exerts a regulatory effect on extracellular levels of agonists at the glycine modulatory site, and consequently on glutamatergic efficacy, with resultant implications for cognitive function and excitotoxic neurodegenerative processes. We propose in this pilot project to clarify the role of the ASCT2 transporter in glutamine export from murine (or alternatively rat) astrocytes using three approaches: (1) We will determine whether the detailed substrate specificity and exchange properties of System ASC-mediated glutamine transport in astrocyte cultures are consistent with the pattern exhibited by the ASCT2 transporter expressed in Xenopus laevis oocytes; (2) We will measure the effect of hybrid depletion of ASCT2, using antisense oligonucleotides, on the velocity of System ASC-mediated glutamine transport in astrocyte cultures; (3) Because ASCT2 expression in astrocyte cultures may not reflect the level of expression in vivo, we will raise in rabbits polyclonal antibodies directed against the ASCT2 transporter in order to detect ASCT2 expression immonocytochemically in astrocytes dissociated acutely from murine brain slices. The results of this pilot project will make possible further investigation of a) the regulation of glutamine export from astrocytes during proliferation, maturation and senescence, and b) the functional implications for the aging brain.